Project Summary The SRY-box2 protein, Sox2, is a dose-dependent master transcriptional factor that regulates the self- renewal and pluripotency or multipotency of embryonic stem cells, many fetal and adult stem cells including neural stem cells. In embryonic stem cells, an increase of Sox2 promotes development into ectoderm and mesoderm lineages, while loss or reduction of Sox2 induces differentiation into endoderm and trophectoderm lineages. Even at the 4-cell embryonic stage, the heterogeneous binding of Sox2 to target genes determines the first lineage decision. Emerging lines of evidence also indicate that gene amplification and over- expression of Sox2 are frequently associated with many cancers. Sox2-expressing cancer cells usually behave as the most primitive cancer progenitor cells that promote hierarchical tumor growth and are usually resistant to various chemo- and radiation therapy. It is clear that the levels of Sox2 should be precisely regulated in development and altered expression of Sox2 can cause various diseases including cancers. However, it remains unclear how the protein stability of Sox2 is dynamically regulated. We have recently found that the protein stability of Sox2 is regulated by a unique and novel methylation-dependent proteolysis in embryonic stem cells. We propose to unravel this important mechanism by identifying the methylation- specific ubiquitin ligase proteins that target the methylated Sox2 protein for degradation. We will also characterize the role of methylation-dependent Sox2 degradation in regulating the self-renewal and pluripotency of embryonic stem cells. Our specific aim 1 is to identify the ubiquitin E3 ligases that target the methylated Sox2 protein for degradation. We hypothesize that the methylated Sox2 is targeted for degradation by specific ubiquitin E3 ligases. We have developed very sensitive assays to identify the ubiquitin E3 ligases for Sox2 degradation. Our specific aim 2 is to determine how the methylated Sox2 protein is recognized and processed for proteolysis. We hypothesize that the methylated lysine residues in Sox2 protein are recognized by specific methyl binding proteins that cooperate with the ubiquitin E3 ligases to target Sox2 for ubiquitin-dependent degradation. We propose to identify these proteins and determine how they specifically recognize the methylated lysine residues in Sox2. Our specific aim 3 is to investigate the physiological roles of the methylated Sox2 degradation in mouse embryonic stem cells. Since Sox2 binds to transcriptional regulatory regions of many important genes to control their gene expression, we propose to monitor whether the dynamic methylation and demethylation of Sox2 protein on chromatin and to determine the role of methylation-dependent degradation of Sox2 during cellular differentiation of embryonic stem cells. Since this is an R15 grant, our proposed research should train graduate students and undergraduate students in stem cell research and help to enhance the research environment for biomedical and stem cell- related research at University of Nevada, Las Vegas.